1. Field of the Invention
The invention relates to an apparatus for confocal observation of a specimen comprising a rotating mask.
2. Description of Related Art
More than 100 years ago the German inventor Paul Nipkow described an imaging method including a plurality of illumination points located on a pinhole mask in such a manner that upon rotation of the pinhole mask around the central rotary axis all points which are to be illuminated are illuminated sequentially—for the same period of time if possible. Such a pinhole mask also is known as “Nipkow disc”. This method enables so-called confocal microscope images by passing the illumination beam path and the image beam path in reverse beam direction through the same pinhole mask, whereby light from outside the object plane can be blocked and whereby good depth resolution can be obtained.
Such a confocal microscope according to the prior art is shown schematically in FIG. 1. The microscope comprises an objective 10 which is for illuminating a specimen 12 in the object plane 14 with illumination light 16 and which collects light reflected from the specimen 12 or emitted from the specimen 12 and images the light—usually by means of a tube lens 18—into the plane of an intermediate image, where a pinhole mask (Nipkow disc) 22 rotating round an axis 20 which is oriented parallel to the beam path is arranged, onto which pinhole disc an appropriate pinhole pattern 24 has been provided which is arranged concentrically around the rotary axis 20. The separation and combination, respectively, of the illumination beam path and the image beam path by using a beam splitter 26 occurs after (when seen from the objective 10) the Nipkow disc/pinhole mask 22, with the light emitted or reflected from the specimen 12 impinging on a detector 28, i.e. being imaged onto the detector 28. Such an arrangement is particularly beneficial for fluorescence microscopy, since in that case illumination/excitation and emission occur at different wavelengths, so that the illumination light and the emission light can be separated from each other essentially without losses, if the beam splitter 26 is dichroic. In the example shown in FIG. 1 the beam splitter 26 transmits the emission light, whereas the illumination light 16 is reflected. With such an arrangement the pinhole mask 22 on the one hand is imaged by the tube lens 18 and the objective 12 into the objective plane 14 and hence onto the specimen 12, while on the other hand the pinhole mask 22 is imaged onto the detector 28, typically by means of two projective lenses 30, by imaging the intermediate image first into infinity and then onto the detector 28. As can be seen in FIG. 1, always only a portion of the area of the Nipkow disc/pinhole mask 22 is illuminated each time.
The desired confocal effect is achieved in that only a small portion of the specimen 12 in the object plane 14 is illuminated at the same time by the illumination light 16, namely only there where a pinhole of the pinhole mask 22 is imaged onto the specimen 12, and in that these illumination points are spaced sufficiently far apart, so that the illumination light 16 passing through the respective pinhole in the pinhole mask 22 is not tampered or tampered only to a small extent by reflected light or emitted light from the specimen 12 caused by an adjacent pinhole of the pinhole mask 22. The pinholes of the pinhole mask 22 hence act as confocal apertures. In a system as shown in FIG. 1 usually only that portion of the excitation light 16 is utilized which passes through the pinholes of the pinhole mask 22, whereas the remaining portion of the illumination light is blocked, so that such systems have a relatively low efficiency and hence have relatively low light transmitting power. An example of such a system can be found in U.S. Pat. No. 6,147,798.
A system and a method allowing for an increased light throughput for a confocal microscope comprising a Nipkow disc is described, for example, in EP 0 539 691 A2. The optical arrangement described there is schematically shown in FIGS. 2 and 3. A microlens arrangement 34 is provided which is axially displaced with regard to the pinhole mask 22 and which has a geometry which is adjusted to the pinhole pattern 24 and which rotates synchronously with the pinhole mask 22 around the same rotary axis 20. Each microlens 36 of the microlens arrangement 34 is located on a disc 38 and serves to concentrate the portion of the illumination light 16 falling onto the respective microlens 36 into the associated pinhole 32 of the pinhole mask 22, i.e. to focus the light onto a focal spot which is located within the respective pinhole 32. Hence, for each pinhole 32 of the pinhole mask 22 there is a conjugate microlens 36 of the microlens arrangement 34. Since the pinhole mask 22 is located in the focal plane of the microlenses 36, the light throughput can be significantly increased thereby, at least if the illumination light beam 16 is a coherent laser light beam. However, one problem of this approach is that beam splitting occurs in the converging beam path between the two discs 22 and 38, where due to the short focal length of the microlenses 36 there is little space for the beam splitter 26, which in this case has to be a short pass filter. In order to avoid image distortions caused by inaccuracies of the surface, the beam splitter 26 must not be too thin. A corresponding thickness of the beam splitter 26, however, creates beam displacement increasing with increasing inclination of the beam splitter 26, which displacement points linearly into the direction of the inclination. For two synchronously rotating discs 22 and 38 having a radially arranged pattern this beam displacement causes that the focal spot of the microlenses 36 does not always coincide with the corresponding pinhole 32 of the pinhole mask 22. This effect has to be compensated by tilting one of the discs relative to the other one.
For a Nipkow disc the pinholes usually are arranged in several spiral tracks or shells which mesh with each other. Since a confocal microscope requires the observation field to be illuminated as homogeneously as possible, all points of the filed have to be illuminated exactly for the same period of time, provided that the pinholes have the same size everywhere and that the illumination is homogeneous. This holds also for the microlenses used. According to EP 0 539 691 A2 homogeneous illumination of the specimen is achieved by keeping constant the tangential distance between adjacent pinholes and by keeping also the radial distance between adjacent shells of the pinholes constant at the same value irrespective of the radius r. Thus, the radial distance between adjacent pinholes varies between the value 1×r and the value 1.12×r, and the filling factor is reduced to a maximum of 78.5% if using circular microlenses are used. Further examples of pinhole patterns for Nipkow discs for microscopes are given in U.S. Pat. No. 5,734,497 and U.S. Pat. No. 5,067,805.
Other confocal microscopes comprising a beam splitter between a Nipkow disc and a microlens arrangement which is axially displaced and which rotates synchronously with the Nipkow disc are described, for example, in US 2007/035734 A1, EP 1 168 029 A2 and US 2003/0215121 A1.
A modified confocal microscope is described in EP 0 753 779 B1, wherein in the region between the microlens disc and the Nipkow disc mirrors are provided in order to pass the light radially out of the space between the two discs for image formation and beam splitting.
In US 2005/094261 A1 a confocal microscope is described, wherein in front of (when seen from the objective) a microlens disc rotating synchronously with the Nipkow disc a second microlens disc is provided, which is axially displaced with regard to the first microlens disc, which likewise rotates synchronously with the Nipkow disc and which is exactly adjusted to the first microlens disc, so as to create an infinity space, i.e. parallel beam paths, between the two microlens discs, in which space the beam splitter is arranged. For this device the mechanical requirements resulting from the demand that the two microlens discs have to be always exactly adjusted to each other are critical.
U.S. Pat. No. 5,760,950 describes a confocal microscope which does not use microlenses and wherein a radial region which is covered by a rotating Nipkow disc, is illuminated with light from a light source, wherein the illumination light having passed through this first region is deflected by 90 degrees by means of a penta prism, and wherein the deflected light is again deflected by 90 degrees by means of a beam splitter and is used for illuminating a second radial region which is covered by the rotating Nipkow disc, which second radial region is located exactly opposite to the rotary axis, i.e. displaced by 180 degrees in the peripheral direction. The second region of the Nipkow disc is imaged onto the specimen by means of the objective, and the light originating from the specimen is spatially filtered by this second region of the Nipkow disc prior to being imaged onto the detector.
It is an object of the invention to provide for a confocal device for observation of a specimen comprising a rotating mask provided with openings, which device should have a light gathering power as high as possible and which nevertheless should have a relatively simple structure.